The objective of this STTR Phase I proposal is to develop novel multi-layer microparticles and demonstrate their feasibility to isolate and expand stem cells for medical research and therapeutic applications. In recent years, stem cells have been used in a wide variety of applications, including cell- based therapies, drug screening, and models to understand the disease development. Unfortunately, many of these applications require large number of stem cells which cannot be produced by most of the current cell isolation and culture methods. The limitations of the current methods include the use of harmful chemicals and enzymes for cell isolation and expansion, and harsh shear forces from centrifugation during cell passaging process. To overcome these drawbacks, we will develop novel multi-layer microparticles that allow stem cell isolation via magnetic separation, cell attachment using a temperature-sensitive mechanism that binds cells at 370C, cell expansion via 3-D structural architecture, and cell detachment by a temperature-sensitive mechanism to release cells bound to the surface at room temperature. These microparticles will provide all-in-one pot for cell isolation, expansion, and detachment of stem cells for use in medical applications without the use of harmful chemicals and mechanical forces. For the phase I study, we will formulate and characterize these multilayer microparticles (Aim 1). The microparticles will then be evaluated for their effectiveness in isolation, expansion, and detachment of stem cells, including endothelial progenitor cells (EPCs) from human blood (Aim 2). The successful completion of this proposal may provide a means to effectively isolate and expand various cell types in addition to stem cells. The knowledge gained from this research should significantly improve our understanding of magnetic cell separation, stem cell expansion, and microparticles for cellular engineering applications.